This invention relates to making insulin-like growth factor (IGF), including IGF-I and IGF-II, in high yield.
Insulin-like growth factors, each with a molecular weight of about 7,500 daltons, possess A and B domains that are highly homologous to the corresponding domains of proinsulin. The A and B domains are connected to each other by a C domain. A carboxy terminal extension, the D domain, is present in IGF but is not found in proinsulin. Both IGF-I and IGF-II are single-chain polypeptides each with 3 disulfide bridges and have a sequence identity of 49% and 47%, respectively, to human insulin A and B chains. Like insulin, IGF stimulate phosphorylation of specific tyrosine residues within the cytoplasmic domain of the receptors to which they bind, as described in WO 93/98826. The designation "insulin-like growth factor" was chosen to express the insulin-like effects and the insulin-like structure of these polypeptides which act as mitogens on a number of cells, as described in EP 128 733. IGF-I is a 70 amino acid peptide, while IGF-II is a 67 amino acid peptide, as described in Rinderknecht, J Biol Chem, (1978) 253:2769; and Rinderknecht, FEBS Letters, (1978) 89:283. IGF-I and IGF-II have 62% structural homology to each other. Both have been isolated from human serum.
Insulin-like growth factors are also known under the class name somatomedins, and have been identified in various animal species as polypeptides that act to stimulate growth of cells in a variety of tissues and cell types, particularly during development. Growth promoting effects of somatomedins include enhancement of cell multiplication and stimulation of cartilage proliferation, stimulation of transport of amino acids, synthesis of RNA, DNA and protein, and stimulation of incorporation of sulfate into proteoglycan and of proline into collagen. Much mammalian postnatal growth is due to stimulation of cartilage growth by somatomedins and growth in utero may also be somatomedin-dependent.
Uses of IGF as a known stimulatory and growth promoting agent includes use for bone repair and replacement therapy, as described in EP 303 855; as a means to counteract certain harmful side effects of carcinostatic drugs, as described in JP 63-196524; and as a way to increase lactation and meat production in cattle and other farm animals, as described in U.S. Pat. No. 4,783,524.
IGF-I has also been found useful in the treatment of osteoporosis in mammals exhibiting decreased cortical bone mineral density and those exposed to drugs or environmental conditions that result in bone density reduction and potentially to an osteoporosis condition, as described in EP 560 723 and EP 436 469.
IGF-I has been administered with sodium pentosan polysulfate (PPS) to severely osteoarthritic canines with the effect of reducing the severity of the disease by lowering the levels of active neutral metalloproteinase in the cartilage. In the model of mildly osteoarthritic canines, therapeutic intervention with IGF-I and PPS together appeared to successfully maintain cartilage structure and biochemistry, while IGF alone was ineffective, as described in Rogachefsky, Osteoarthritis and Cartilage, (1993) 1:105-114. Recombinant proteins have been made by many processes known in the art, as described in U.S. Pat. No. 4,870,008, EP 324 274, EP 123 228, EP 501 914, EP 128 733, WO 93/11240, EP 135 094, U.S. Pat. No. 5,158,875, WO 85/00831, EP 264 074, EP 219 814, EP 155 655, EP 379 338, U.S. Pat. No. 4,876,242, U.S. Ser. No. 578,728, U.S. Ser. No. 747,152, U.S. Pat. No. 4,963,665, U.S. Ser. No. 681,688, U.S. Ser. No. 837,313, WO 89/03423, U.S. Pat. No. 5,210,028, and U.S. Pat. No. 5,231,178.
Moreover, IGF can be produced in methylotrophic yeast transformants with the IGF coding sequence linked to a signal sequence which direct secretion and proteolytic processing of the protein product. The signal sequence suitable herein includes the S. cerevisiae alpha mating factor pre-pro sequence in protease deficient P. pastoris strains, as described in WO 92/04363.
Recombinant IGF can be grown and harvested in favorable growth conditions as described in U.S. Pat. No. 4,810,646 and U.S. Pat. No. 4,992,540.
IGF-I and IGF-II have been expressed and secreted using a leader sequence that contains a portion of the yeast .alpha.-factor signal sequence, as described in EP 128 733.
It would be advantageous to produce IGF in high yield using a Saccharomyces host cell.